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Patrick J. Halbach, Matti Asuma, Weinxiang Luo, De-Ann M. Pillers, Bikash R. Pattnaik; Oxytocin Regulation of Retinal Pigment Epithelium Cell Function. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5384.
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Oxytocin (OT) is a neuropeptide that activates oxytocin receptor (OTR), a rhodopsin family G-protein coupled receptor. OTR signals the cellular phosphatidylinositol-calcium second messenger system. Within the retina, the cross talk between retinal pigment epithelium (RPE) and photoreceptor neurons is hypothesized to be mediated by one such phosphatidylinositol-calcium signaling mechanism. Several molecules, particularly ATP, are proposed to mediate RPE-retina communication. Since we have localized OTR transcripts to RPE, we were interested in knowing whether RPE cells also utilize oxitocinergic signaling for communication.
Human fetal RPE (hRPE) cells were cultured as a tight monolayer using serum-free medium containing MEM alpha base medium, N1 supplement, non-essential aminoacids, taurine, hydrocortisone and triiodo-thryonin. Cultures were maintained at 37°C and 5% CO2 with a media change every 2-3 days. Cytoplasmic content of isolated cells were harvested and analyzed by single-cell RT-PCR. We used a two-step nested PCR method to amplify the transcripts. Immomix PCR mix and appropriate primer pairs were utilized to amplify OTR and other RPE-specific transcripts in individual cells. Localization of OTR to the RPE layer of monkey frozen sections was determined by standard immunofluorescence methods. Intracellular Ca2+ ([Ca2+]i) mobilization in response to OT and ATP was conducted via live-cell imaging and FURA-2 ratiometric measurements.
In four individual hRPE cells, a 256 bp transcript corresponding to the OTR messenger was amplified. These cells also tested positive for RPE-specific markers RPE65, Kir7.1, hBest1, NaKATPase, and ezrin transcripts. OTR was localized to the posterior aspects of monkey RPE cells. hRPE cells in culture, when stimulated by OT or ATP, exhibited a 70-120 nM increase of [Ca2+]i that was completely reversible. However, while ATP induced an instantaneous increase in Ca2+, OT induced a gradual rise, suggesting ATP and OT may be acting through independent signaling mechanisms.
Localization of OTR in RPE is a novel finding that implicates an important regulatory role of OT in the RPE-retina communication. The increase of [Ca2+]i in response to OT stimulation, as well as previously established circadian regulation of the RPE, suggests that the RPE may utilize oxytocinergic signaling as one aspect of endocrine regulation of RPE function.
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